PSA-NCAM is up-regulated during optic nerve regeneration in lizard but not in goldfish.

The addition of polysialic acid (PSA) to neural cell adhesion molecule (NCAM) facilitates axon growth. Here we use Western blots and immunohistochemistry to examine expression of PSA-NCAM during optic nerve regeneration. In lizard, retinal ganglion cell axons become transiently PSA-NCAM positive. By contrast, goldfish RGC axons are PSA-NCAM negative both in normal animals and throughout regeneration with the exception of a PSA-NCAM-positive fascicle arising from newly generated RGCs. Transient sialylation of NCAM in lizard may assist regeneration in the nonpermissive reptilian visual pathway and facilitate the reestablishment of a crude topographic map; down-regulation in the long term may contribute to the breakdown in topography. The lack of sialylation in goldfish presumably reflects the permissive nature of the substrate allowing axon regeneration and the successful reestablishment of a topographic map.

Continued neurogenesis is not a pre-requisite for regeneration of a topographic retino-tectal projection.

Electrophysiological recording demonstrated that visuo-tectal projections are topographically organised after optic nerve regeneration in aged Xenopus laevis. 3H-thymidine autoradiography confirmed previous reports [Taylor, Lack, & Easter, Eur. Journal of Neuroscience 1 (1989) 626-638] that cell division had already ceased at the retinal ciliary margin. The results demonstrate that, contrary to a previous suggestion [Holder & Clarke, Trends in Neuroscience 11 (1988) 94-99], continued neurogenesis is not a pre-requisite for the re-establishment of appropriate connections with target cells.

Evidence that regenerating optic axons maintain long-term growth in the lizard Ctenophorus ornatus: growth-associated protein-43 and gefiltin expression.

In the lizard, Ctenophorus ornatus, the optic nerve regenerates but animals remain blind via the experimental eye, presumably as a result of axons failing to consolidate a retinotopic map in the optic tectum. Here we have examined immunohistochemically the expression of the growth-associated protein GAP-43 and the low-molecular-weight intermediate filament protein gefiltin, up to one year after optic nerve crush. Both proteins were found to be permanently up-regulated, suggesting that regenerating axons are held in a permanent state of re-growth. We speculate that, in the lizard, the continued expression of GAP-43 and the failure to switch from the expression of low- to high-molecular-weight intermediate filament proteins are associated with the inability to consolidate a retinotopic projection.

The relationship between the position of the retinal area centralis and feeding behaviour in juvenile black bream Acanthopagrus butcheri (Sparidae: Teleostei).

The topography of the neurons in the retinal ganglion cell layer of juvenile black bream Acanthopagrus butcheri changes during development. The region of high cell density the area centralis (AC), relocates from a temporal (central) to a dorsal (peripheral) position within the dorso-temporal retinal quadrant. To ascertain whether the differences in the position of the AC during development are related to feeding behaviour, we monitored fishes that were given a choice of food. A range of feeding behaviour patterns was recorded in individual fishes. The smallest fishes (8-15 mm standard length (SL)) took live food from the water column. Following weaning onto pellets, fishes exhibited a preference for taking food from either the substrate or the surface (but not both). When greater than 20 mm SL, a number of individuals then divided their time between surface and substrate feeding before all fishes became exclusive benthic feeders at a stage between 50 and 80 mm SL. Three individual fishes, for which behaviour patterns were categorized, were killed and the topography of the retinal ganglion cell layer analysed. A range of positions for the AC was found with the smallest fish (12 mm SL) possessing a region of high cell density in the temporal retina. In a larger fish (70 mm SL), feeding from both the substrate and the surface, the AC was found in an intermediate dorso-temporal position. The AC of a fish (51 mm SL) preferentially taking food from the substrate was located in a dorsal position.

Variability in the location of the retinal ganglion cell area centralis is correlated with ontogenetic changes in feeding behavior in the black bream, Acanthopagrus butcheri (Sparidae, teleostei).

The development of neural cell topography in the retinal ganglion cell layer was examined in a teleost, the black bream (Acanthopagrus butcheri). From Nissl-stained wholemounts, it was established that fish between 10 and 15 mm standard body length (SL) possess high cell densities throughout the dorso-temporal retinal quadrant, with peak cell densities located in temporal regions of the retina. However, in fish between 15 and 80 mm SL, a wide variation in the position of the peak cell density is revealed with the locations of the areae centrales (AC) ranging from exclusively temporal to periphero-dorsal retina. Fish larger than 80 mm SL always possess an AC located in the dorsal region of the dorso-temporal retinal quadrant. The topography of ganglion cells within the ganglion cell layer was determined by comparing the numbers of ganglion cells retrogradely-labeled from the optic nerve with the total population of Nissl-stained neurons (ganglion plus displaced amacrine cells) in a range of different-sized individuals. Ganglion cell topography was the same as that recorded for all Nissl-stained neurons. The feeding behavior of juveniles from metamorphosis to 80 mm SL was observed, where fish were given the choice of feeding on live food in mid-water (until 15 mm SL) or obtaining pellets from the surface or the bottom. A range of feeding patterns was recorded, with the smallest fish taking food from mid-water but individuals between 15 and 80 mm SL taking food either from the surface or the bottom or both. A correlation between the preferred mode of feeding and the position of the AC was found, such that those individuals feeding in mid-water or at the surface possess a temporal or intermediate (dorso- temporal) AC, whereas those predominantly feeding from the bottom possess a dorsal AC.

Horse vision and an explanation for the visual behaviour originally explained by the 'ramp retina'.

Here we provide confirmation that the 'ramp retina' of the horse, once thought to result in head rotating visual behaviour, does not exist. We found a 9% variation in axial length of the eye between the streak region and the dorsal periphery. However, the difference was in the opposite direction to that proposed for the 'ramp retina'. Furthermore, acuity in the narrow, intense visual streak in the inferior retina is 16.5 cycles per degree compared with 2.7 cycles per degree in the periphery. Therefore, it is improbable that the horse rotates its head to focus onto the peripheral retina. Rather, the horse rotates the nose up high to observe distant objects because binocular overlap is oriented down the nose, with a blind area directly in front of the forehead.

Residual vision in a subject with damaged visual cortex.

It is well known that a lesion in the optic radiation or striate cortex leads to blind visual regions in the retinotopically corresponding portion of the visual field. However, various studies show that some subjects still perceive certain stimuli even when presented in the "blind" visual field. Such subjects either perceive stimuli abnormally or only certain aspects of them (residual vision) or, in some cases, deny perception altogether even though visual performance can be shown to be above chance (blindsight). Research on monkeys has suggested a variety of parallel extrastriate visual pathways that could bypass the striate cortex and mediate residual vision or blindsight. In the present study, we investigated a subject with perimetrically blind visual areas caused by bilateral brain damage. Black and white stimuli were presented at many locations in the intact and affected areas of the visual field. The subject's task was to state, using confidence levels, whether the target stimulus was black or white. The results revealed an area in the "blind" visual field in which the subject perceived a light flash when the experimental black stimulus was presented. We hypothesize that a spared region in the visual cortex most likely accounts for these findings.

Number of neurons in the retinal ganglion cell layer of the quokka wallaby do not change throughout life.

During adult life, the topography of the retinal pigment epithelium (RPE) of the quokka wallaby changes gradually. Cells in peripheral retina enlarge in surface area while those in mid-temporal retina, adjacent to the area centralis, a high density region in the ganglion cell layer, decrease in area, implying that the tissue in this area is drawing together. We speculated that high ganglion cell densities in temporal regions might be maintained, in the face of cell loss due to aging, by this apparent drawing together of the RPE sheet. Therefore, we examined the retinal ganglion cell layer of the quokka in cresyl violet stained wholemounts from animals aged from 0. 55 to 13.5 years. We found that total neuron number in the retinal ganglion cell layer of the quokka did not decrease significantly throughout life even though individuals in captivity live long lives (9-15 years). Ganglion and amacrine cells were counted separately and identified by strict morphological criteria. Nevertheless, the proportion of ganglion to amacrine cells appeared to decrease linearly throughout life, indicating that the morphology of a proportion of neurons became more amacrine-like during aging. Mean cell size did not change throughout life. In the quokka, retinal area increases slowly throughout life and may account for the small reduction in cell density seen in most retinal regions.

Experimental eye enlargement in mature animals changes the retinal pigment epithelium.

Form deprivation has been shown to result in myopia in a number of species such that the eye enlarges if one eye is permanently closed at the time of eye opening. In the quokka wallaby, the eye grows slowly throughout life. After form deprivation, the eye enlarges by 1-1.5 years of age to the size of that in a 4-6-year-old animal and the number of multinucleated retinal pigment epithelial (RPE) cells in the enlarged retina remains much lower than would be expected in eyes of comparable size. Here we have repeated the experiment but examined animals at 4 years of age. The sutured eye grew significantly larger than did its partner. Numbers of RPE cells were comparable between sutured and partner eyes but were lower than in normal animals of similar age. Reductions in RPE cell density were greater in nasal than in dorsal or ventral retina and were not seen in temporal retina. The distribution of multinucleated cells was quite different in the sutured and open eyes. As in normal eyes, partner eyes had most multinucleated cells in ventral retina, while in the sutured eyes such cells were located mainly in the far periphery. In conclusion, the RPE is significantly changed by the eye enlargement process. However, it is not known whether this change results from an active part played by the RPE in the retinal expansion process or whether the changes are simply a result of a passive increase in area of the RPE.

Retinal structure and visual acuity in a polyprotodont marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata).

The visual system of the fat-tailed dunnart (Sminthopsis crassicaudata), a small polyprotodont marsupial, has been examined both anatomically and behaviourally. The ganglion cell layer was examined in cresyl-violet stained wholemounts and found to contain a mean of 81,400 ganglion cells (SD +/- 3,360); the identification of ganglion cells was supported by a correspondence to optic axon counts. Ganglion cells were distributed as a mid-temporally situated area centralis, embedded in a pronounced visual streak. Localised implants of horseradish peroxidase into retinal wholemounts revealed both A-type and B-type horizontal cells. Sections of the outer retina showed it to be rod-dominated, with a rod-to-cone ratio of 40:1 at the area centralis; cones were found to contain oil droplets but double cones were not a prominent feature. The retinal pigment epithelium consisted of squamous cells. Visual acuity, estimated from counts of peak ganglion cell density (8,300/mm2, SD +/- 1,180) and measurements of posterior nodal distance (2.9 mm), was found to be 2.30 cycles per degree. The value was close to that of 2.36 cycles per degree estimated by behavioural tests using a Mitchell jumping stand; values were similar at low, intermediate and high light levels. Our findings are discussed in relation to the lifestyle of the dunnart.

Blindsight in subjects with homonymous visual field defects.

Brain damage in the visual system can lead to apparently blind visual areas. However, more elaborate testing indicates that some visual ability may still exist for specific stimuli in the otherwise blind regions. This phenomenon is called 'blindsight' if subjects report no conscious awareness of visual stimuli but when forced to guess, nevertheless perform better than chance. It has mainly been suggested that secondary visual pathways are responsible for this phenomenon. However, no published study has clearly shown the neural mechanism responsible for blindsight. Furthermore, experimental artifacts may have been responsible for the appearance of the phenomenon in some subjects. In the present study, the visual fields of nine subjects were mapped and residual visual performance was examined in many areas using three different experimental procedures. Artifacts such as stray light or eye movements were well controlled. In addition, confidence ratings were required after each trial in the forced-choice tests. The results show that only one subject with a lesion in the optic radiation had blindsight in two discrete areas of the affected visual field. Spared optic radiation fibers of the main (primary) geniculo-striate visual pathway were most likely to account for this finding.

The effects of a transient increase in temperature on cell generation and cell death in the hippocampus and amygdala of the wallaby, Setonix brachyurus (quokka).

We examined the effects of a single exposure to a temperature elevation of 2 degrees C for a 2 h period on the developmental processes of cell division and cell death in the hippocampus and the amygdala of the quokka. Animals aged postnatal day (P) 4045 were injected with tritiated (3H-) thymidine and then exposed to either 37 degrees C (normal) or 39 degrees C (+/-0.2 degrees C) in an incubator for a duration of 2 h. The young were then returned to the nipple and, after a period of 24 h, were sacrificed. Brains were sectioned and selected sections processed for autoradiography, and some were counterstained. Cell division taking place at the time of heating was estimated by counting 3H-thymidine-labelled cells and at the time of sacrifice by counting mitotic figures. Dying cells were visualised as pyknotic profiles in cresyl-violet-stained sections. In both the amygdala and the hippocampus, the number of 3H-thymidine-labelled cells (cells dividing in situ during the heating period) was significantly lower in the experimental than the control group. Such cells were glia in the amygdala and granule cells and glia in the hippocampus. However, the number of dying cells or mitotic figures (cells dividing at the time of sacrifice) did not differ significantly between the two groups. By comparison, the number of 3H-thymidine-labelled cells, dying cells or mitotic figures did not significantly differ in the diencephalon. Therefore, a brief exposure to a slight elevation in temperature results in an immediate alteration in cell division in the hippocampus and amygdala. These findings have implications for the role played by raised temperature, such as during virus infection, in producing developmental anomalies of the brain.

Anatomical comparison of the macaque and marsupial visual cortex: common features that may reflect retention of essential cortical elements.

This study identifies fundamental anatomical features of primary visual cortex, area V1 of macaque monkey cerebral cortex, i.e., features that are present in area V1 of phylogenetically distant mammals of quite different lifestyle and features that are common to other regions of cortex. We compared anatomical constituents of macaque V1 with V1 of members of the two principal marsupial lines, the dunnart and the quokka, that diverged from the eutherian mammalian line over 135 million years ago. Features of V1 common to both macaque and marsupials were then compared with anatomical features we have previously described for macaque prefrontal cortex. Despite large differences in overall area and thickness of V1 cortex between these animals, the absolute size of pyramidal neurons is remarkably similar, as are their specific dendritic branch patterns and patterns of distribution of intrinsic axons. Pyramidal neuron patchy connections exist in the supragranular V1 in both the marsupial quokka and macaque as well as in macaque prefrontal cortex. Several specific types of aspinous interneurons are common to area V1 in both marsupial and macaque and are also present in macaque prefrontal cortex. Spiny stellate cells are a common feature of the thalamic-recipient, mid-depth lamina 4 of V1 in all three species. Because these similarities exist despite the very different lifestyles and evolutionary histories of the animals compared, this finding argues for a highly conserved framework of cellular detail in macaque primary visual cortex rather than convergent evolution of these features.

Development and aging of cell topography in the human retinal pigment epithelium.

PURPOSE: To determine how regional cell density of this tissue changes with age, the authors examined the topography of the human retinal pigment epithelium (RPE) in wholemounted tissue obtained from eyes aged 12 to 89 years, donated for corneas. METHODS: The RPE, with choroid attached, was wholemounted and stained with cresyl violet. From these preparations, the authors analyzed retinal area, RPE cell number, and cell density. RESULTS: Retinal pigment epithelial cell number is highly variable between persons but does not appear to be age related. Retinal area increases until approximately 30 years of age, but beyond this age individual variation masks further enlargement. The distinctive topography of the RPE changes markedly with age. There is a modification from the relatively homogeneous cell density distribution in the youngest retinas examined toward a more heterogeneous pattern in older retinas. From mid-adolescence, a band of larger cells appears at the extreme periphery, adjacent to the ora serrata, which gradually widens so that by 90 years of age, it occupies the outermost 30% of the retinal area. Cell density is highest in the central temporal retina, adjacent to the macula in the neural retina, throughout life. Cell density values in this region increase slightly with age, and the difference between this and surrounding regions becomes more marked with age. CONCLUSIONS: With no marked change in total cell number, peripheral RPE in humans enlarges in area throughout life, but the RPE in more central regions decreases in area.

Changing topography of the RPE resulting from experimentally induced rapid eye growth.

The retinal pigment epithelium (RPE) of the quokka wallaby. Setonix brachyurus, grows and changes throughout life. To investigate factors that determine changes in the quokka RPE, we have examined topography of this tissue in experimentally enlarged eyes. Unilateral eyelid suture was conducted at the time of normal eye opening, postnatal day (P) 110, and animals were examined at 1 or 1 1/2 years of age. The numbers and densities of RPE cells and the extent of multinucleation were compared with those in normal animals. Eyelid suture resulted in a 9.8% and 17.4% increase in retinal area at 1 and 1 1/2 years, respectively; a significant degree of myopia was associated with this enlargement. Cell density topography in experimental eyes was not the same as in controls. Cells from central retina were disproportionately larger in the experimental than control eyes. However, the RPE cell topography in sutured eyes was not the same as that of aged retinae of a similar size. Notably, in sutured eyes there was no development of the high or highest cell densities seen in equatorial and temporal central RPE in aged retinae, respectively. Furthermore, the degree of cell enlargement in peripheral regions was slight compared with that observed in similar-sized, aged retinae. There was no increase in RPE cell number; rather, average cell area increased accompanied by no change or a slight decrease in RPE thickness. Consequently, overall volume of cells did not change significantly. The large number of multinucleate cells normally seen in aged animals was not observed in experimentally enlarged eyes, implying that an increase in cell volume may be the trigger for multinucleation.

Development and cell generation in the hippocampus of a marsupial, the quokka wallaby (Setonix brachyurus).

Development and cell generation in the hippocampus of the marsupial, the quokka wallaby, has been examined. Cells in this brain region are similar in morphology to those in eutherian species, with predominantly pyramidal and granule cells. In the quokka, development of the hippocampus takes place postnatally; this region is first seen just after birth on postnatal day 1 (P1) as an out-pouching of the medial cortical wall into the lateral ventricle. The cornu ammonis (CA) region first appears at P20 as a line of denser cells and by P30, CA3 and the granule cell layer of the dentate gyrus (DG) can be defined. A specific region of the ventricle, near to the developing fimbria, produces the granule cells destined for the dentate gyrus. These cells initially migrate in a curved trajectory into the hilus, following the path of thick, vimentin-positive glial fibres. Cells are generated in the hippocampus from around P5 until at least P85 when some cells in the hilus and also glial cells are labelled with [3H]thymidine. In the cell sparse region around the hippocampal fissure there is a peak of neuron production before P20 followed by a decline and subsequent increase in the production of probably glial cells after P60. The peak of cell generation in the CA region and the granule cell layer of the DG is around P40. Cells continue to be produced in the hilus of the DG much later, with numbers still high at P85, presumably these cells are destined to reach the granule cell layer later in development.

Retinal pigment epithelium and photoreceptor maturation in a wallaby, the quokka.

Cell generation and the early stages of maturation of the retinal pigment epithelium (RPE) and photoreceptors were examined in a marsupial, the quokka, Setonix brachyurus. Results are presented for animals aged up to postnatal day (P)250. RPE cell generation was studied by analysis of cell number from wholemounted retinae and by tritiated thymidine (3HThy) autoradiography in sectioned material. For 3HThy autoradiography, quokkas aged P1-P200 were injected with 3HThy and killed either 6-20 hours later (pulse-kill) or at P100 or P250 (pulse-leave). The extent of pigmentation of the RPE sheet was examined from sections of embryonic and early postnatal stages. Retinae from animals aged P5 to P160 were also examined at the electron microscope. By P100, RPE cell number is within the range found in adults. New RPE cells are generated in a peripheral band which moves outwards as cells leave the cell cycle in more central locations. RPE cells thus complete their last cell division in a centre-to-periphery wave centred about the optic nerve head. At any given retinal location, RPE cells complete their last cell division earlier than the overlying layers of the neural retina. Cells of the RPE rapidly develop a mature morphology. For example, melanin granules are observed at P5 and Verhoeff's membrane (the terminal bar complex) is evident by P25. By contrast, photoreceptor development in this species is protracted; cone inner segments are observed by P40, whilst the first rod inner segments are observed at P60. Despite being generated earlier, morphological maturation of the cones appears retarded and prolonged compared with that of the rods. The last stages of RPE cell maturation occur late in development, in synchrony with the generation of rods.

Development and ageing of the RPE in a marsupial, the quokka.

We have previously shown that the mature adult quokka, aged between 8 and 15 years, has a distinct cell topography in the retinal pigment epithelium (RPE). We reported that the adult cell densities were high in central temporal retina and low in a peripheral band, adjacent to the ora serrata, a region with a concentration of multinucleate cells. In the present paper, we have studied the development of these features in order to understand how they mature, as well as to gain insight into regional specializations of the RPE. Retinal area, cell density and the extent of multinucleation were analysed using whole-mounted retinae from animals aged post-natal day (P) 2 to 15. The retina continues to grow in area throughout life, however, RPE cell number does not change. The features of the mature adult RPE develop at different times over the entire lifespan of the animal. In peripheral retina, cell density decreases throughout life and the band of low cell density becomes progressively wider and more distinct with age with an increasing proportion of multinucleate cells. By contrast, RPE cell density in equatorial retina remains, throughout life, at the level observed in 1-year-old animals. A specialization of high cell density in temporal central RPE was discernible in animals older than 2 years, with the cell density of this region increasing steadily beyond this age. Central regions of other quadrants demonstrate a constant and relatively uniform density with age. The RPE in the marsupial quokka is a dynamic tissue, demonstrating topographic changes throughout life.

Retinal pigment epithelium topography in the mature quokka, Setonix brachyurus.

In this paper we describe cell topography of the retinal pigment epithelium (RPE) for the mature marsupial wallaby, the quokka, Setonix brachyurus. RPE topography was analysed in bleached and stained whole mounted retinae, sampling from the entire surface area. The mature adult quokka RPE has a distinct topography in terms of both cell density and the distribution of multinucleate cells. Peripheral RPE demonstrates the lowest cell density and the greatest proportion of multinucleate cells. In an annulus surrounding central retina, corresponding to equatorial retina, RPE cell density is relatively high and multinucleate cells are at the lowest frequency. Cell density is highest in central temporal retina, in the regions adjacent to some of the highest densities in the neural retina. Other regions of central retina exhibit moderate cell densities. A small proportion of central cells are multinucleate. The RPE topography may result from, or account for, regional differences in susceptibility of this tissue to environmental influences and stressors. Understanding of this topography may throw light on the marked localization of certain human retinal diseases inherent to this tissue.

Development of the chiasm of a marsupial, the quokka wallaby.

We have previously shown that the mature optic chiasm of a marsupial is divided morphologically into three regions, two lateral regions in which ipsilaterally projecting axons are confined and a central region containing only contralaterally projecting axons. By contrast, in the chiasms of eutherian (placental) mammals studied to date, there is no tripartite configuration. Ipsilaterally and contralaterally projecting axons from each eye are mixed in the caudal nerve and in each hemichiasm and encounter axons from the opposite eye near the midline of the chiasm. Here, we show that, unlike eutherians, marsupials have astrocytic processes in high concentrations in lateral regions of the nerve and rostral chiasm. Early in development, during the period when optic axons are growing through the chiasm, many intrachiasmatic cells are seen with densities five to eight times higher in lateral than in central chiasmatic regions. Such cells continue to be added to all chiasmatic regions; later in development, considerably more are added centrally, as the chiasm increases in volume. In the mature chiasm, cell densities are similar in all regions. By contrast to the marsupial, cell addition in the chiasm of a placental mammal, the ferret, is almost entirely restricted to later developmental stages, after axons have grown through the chiasm, and there are no obvious spatial variations in the distribution of cells during the period examined. During development, similar to the adult marsupial, ipsilaterally projecting axons do not approach the chiasmatic midline but remain confined laterally. We propose that the cells generated early and seen in high densities in the lateral chiasmatic regions of the marsupial may play a role in guiding retinal axons through this region of pathway selection. These data suggest that there is not a common pattern of developmental mechanisms that control the path of axons through the chiasm of different mammals.